The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) central to metabolic regulation, and its potential modulation by dietary phytochemicals is increasingly recognized as physiologically relevant. Understanding how such compounds interact with GLP-1R is important for clarifying mechanisms that may contribute to gut-to-brain signaling. In this study, we examined three structurally related dietary ginsenosides, Rg1, Rg2, and Rg3, as potential modulators of GLP-1R using luciferase reporter assays and computational analyses. Despite sharing similar molecular weights, a common dammarane scaffold, and comparable sugar moieties, the three ginsenosides displayed distinct effects on GLP-1R activity: Rg2 and Rg3 potently reduced receptor activation in a dose-dependent manner when co-administered with Exendin-4, whereas Rg1 had minimal effect. Computational screening of the GLP-1R structure for binding sites identified a putative extracellular pocket on the protein that can accommodate these compounds, while molecular docking and binding free energy calculations provided predicted affinities qualitatively reflecting the phytochemicals’ experimental activities. These findings point to a plausible extracellular mechanism through which dietary ginsenosides may influence GLP-1R responsiveness at the intestinal interface. Our results point to the possibility that non-absorbed phytochemicals can differentially modulate gut-expressed receptors, suggesting a novel pathway for dietary signaling relevant to ethnopharmacology and metabolic health.

By-products (fly ash) obtained from the combustion process of boiler fuels (coal) is a valuable asset to the construction industry. In order to control the pollution, industries are adopting cofiring of coal with bio-fuels. These cofired ashes alter in physical and chemical properties with reference to (a by-product of burnt coal) fly ash. Detailed study of the potential raw material and its end application as a novel construction material is reviewed. Co-fired ash properties are dominated by biomass type and its proportion in the blend. Critical study of co-fired ash as a supplementary cementitious material and during the production of blended cement has been reviewed with reference to relevant standards. The article discusses the effect of co-fired fly ash inclusion in concrete properties (heat of hydration, workability, setting time, compressive strength, water absorption, freezing and thawing, alkali silica reaction etc.).

The hypothalamic neuropeptide Gonadotropin-Releasing Hormone (GnRH) is one of the key modulators of gonadotropin release and thereby of reproductive development. Unlike in humans, in fish, the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are secreted by two distinct cell populations, which in tilapia also express two types of GnRH receptors, namely tiGnRH-R1 (tiFSHRH-R) and tiGnRH-R3 (tiLHRH-R), respectively. This study characterizes the structural differences between tiFSHRH-R and tiLHRH-R and their human homolog and analyzes their binding sites and the docking of the GnRH decapeptide to each receptor. Interestingly, major differences were observed in GnRH docking conformation, as it is bound to tiLHRH-R in horizontal- and linear conformation when bound to tiFSHRH-R. This conformation allows the hormone to better interact with the extracellular loops and the V-shaped indention on the extracellular domain of the receptors, which play a crucial role in receptor activation, thus providing a possible explanation for previously observed different activation responses. Molecular dynamics simulation showed that despite undergoing larger post-docking conformational changes, the tiLHRH-R-cGnRH complex seemed to trend towards a more stabilized conformation comparatively more quickly than the tiFSHRH-R-cGnRH complex, in which the ligand underwent major positional changes. Finally, we have developed GnRH analogs and super-analogs based on cGnRH and sGnRH deca-peptides, respectively, and showed that they are more potent agonists that also display high specificity for either tiGnRH3 or tiGnRH1.

In brief:The hypothalamus-pituitary-gonad axis integrates environmental and internal signals to tune reproductive functions. We hypothesized that in fish, Gnrh predominantly stimulates luteinizing hormone (LH) secretion, while Cck selectively regulates follicle-stimulating hormone (FSH), thereby uncovering a novel role for Cck as a metabolic gatekeeper that links reproductive activity with nutritional status. Abstract:In fish, the hypothalamus-pituitary-gonad axis governs reproduction in response to environmental and internal signals. FSH regulates gonadal growth, while LH controls maturation. Gonadotropin-releasing hormone (Gnrh) plays a key role in this process, while the role of the satiety hormone cholecystokinin (Cck) is under investigation. We hypothesized that Gnrh and Cck differentially regulate tilapia LH and FSH, reflecting distinct reproductive and metabolic roles. Gnrh receptor (r) 1 was localized to FSH cells and detected only in juvenile fish, while GnRrhr3 was found in LH cells across both juvenile and mature stages. GnRH stimulation significantly increased LH release, while FSH levels showed only a moderate rise. Structural analysis revealed that Gnrhr3 exhibits more stable and rapid ligand binding and activates protein kinase A and protein kinase C (PKC) pathways, whereas Gnrhr1 signals exclusively through the PKC/Ca2+ pathway. Elevated FSH levels and gnrhr 1 expression were observed during early vitellogenesis, coinciding with increased cck-rba expression in FSH cells. Cck-positive neurons originating in the brain were observed to terminate in the adenohypophysis, specifically near FSH-secreting cells. In addition, c ck-rba was exclusively expressed in FSH cells, and tilapia Cck injection significantly elevated plasma FSH levels while reducing food intake, highlighting the role of Cck in linking reproduction to nutritional status. Together, these findings indicate that Gnrh predominantly regulates LH secretion via Gnrhr3, while Cck, through Cck-rba, specifically controls FSH, linking reproduction to nutritional status. Cck acts as a metabolic gatekeeper, aligning gonadal development with energy availability and ensuring reproduction when energy is sufficient.

Surge release of luteinizing hormone (Lh) from the pituitary is essential for fertility as it triggers ovulation. While secretoneurin (SN) is a phylogenetically conserved secretogranin-2-derived peptide that stimulates Lh, its role in ovulation has not been established. To directly compare periovulatory changes in the classical hormones to the emerging reproductive neuropeptides SNa and SNb, simultaneous mass spectrometry measurement of 9 peptides and 5 steroids was conducted in female zebrafish. Regression analysis indicated that levels of SNa1–34 in the brain peaked when type 3 gonadotropin-releasing hormone (Gnrh3) increased (R2 = 0.71) at the time of the Lh surge, 3.5 h before ovulation. Levels of the naturally occurring derivative SNa1–14 were highest at ovulation, while SNb1–31 was invariable. The bioactivities of SNa1–34 and SNa1–14 were investigated. After injection of SNa1–34 in females that had been isolated from males, 61% (11/18) ovulated within 6 h, which was like the effects of the Lh analog human chorionic gonadotropin (72%; 13/18 females). SNa1–34 injection induces ovulation by increasing time-dependent expression of gnrh3 in the brain, a likely direct stimulation of chorionic gonadotropin alpha (cga) and luteinizing hormone b (lhb) subunit in pituitary, and via the subsequent time-dependent increase in nuclear progesterone receptor (npr) in ovaries. In contrast, SNa1–14 exhibited far fewer effects on gene expression and did not induce ovulation. Our results support the proposal that SN is a reproductive hormone.

The manipulation of the somatotropic axis, governing growth, has been a focus of numerous transgenic approaches aimed at developing fast-growing fish for research, medicine and aquaculture purposes. However, the excessively high growth hormone (GH) levels in these transgenic fish often result in deformities that impact both fish health and consumer acceptance. In an effort to mitigate these issues and synchronize exogenous GH expression with reproductive processes, we employed a novel transgenic construct driven by a tilapia luteinizing hormone (LH) promoter. This approach was anticipated to induce more localized and lower exogenous GH secretion. In this study, we characterized the growth and reproduction of these transgenic LHp-GH zebrafish using hormonal and physiological parameters. Our findings reveal that LHp-GH fish exhibited accelerated growth in both length and weight, along with a lower feed conversion ratio, indicating more efficient feed utilization, all while maintaining unchanged body proportions. These fish demonstrated higher expression levels of LH and GH in the pituitary and elevated IGF-1 levels in the liver compared to wild-type fish. An examination of reproductive function in LHp-GH fish unveiled lower pituitary LH and FSH contents, smaller follicle diameter in female gonads, and reduced relative fecundity. However, in transgenic males, neither the distribution of spermatogenesis stages nor sperm concentrations differed significantly between the fish lines. These results suggest that coupling exogenous GH expression with endogenous LH expression in females directs resource investment toward somatic growth at the expense of reproductive processes. Consequently, we conclude that incorporating GH under the LH promoter represents a suitable construct for the genetic engineering of commercial fish species, providing accelerated growth while preserving body proportions.

Somatostatin (SST) plays diverse physiological roles in vertebrates, particularly in regulating growth hormone secretion from the pituitary. While the function of SST as a neuromodulator has been studied extensively, its role in fish and mammalian reproduction remains poorly understood. To address this gap, we investigated the involvement of the somatostatin system in the regulation of growth and reproductive hormones in tilapia. RNA sequencing of mature tilapia brain tissue revealed the presence of three SST peptides: SST6, SST3, and low levels of SST1. Four different isoforms of the somatostatin receptor (SSTR) subfamily were also identified in the tilapia genome. Phylogenetic and synteny analysis identified tiSSTR2-like as the root of the tree, forming two mega clades, with SSTR1 and SSTR4 in one and SSTR2a, SSTR3a, and SSTR5b in the other. Interestingly, the tiSSTR-5 isoforms 5x1, 5x2, and 5x3 were encoded in the sstr3b gene and were an artifact of misperception in the nomenclature in the database. RNA-seq of separated pituitary cell populations showed that SSTRs were expressed in gonadotrophs, with sstr3a enriched in luteinizing hormone (LH) cells and sstr3b significantly enriched in follicle-stimulating hormone (FSH) cells. Notably, cyclosomatostatin, an SSTR antagonist, induced cAMP activity in all SSTRs, with SSTR3a displaying the highest response, whereas octreotide, an SSTR agonist, showed a binding profile like that observed in human receptors. Binding site analysis of tiSSTRs from tilapia pituitary cells revealed the presence of canonical binding sites characteristic of peptide-binding class A G-protein-coupled receptors. Based on these findings, we explored the effect of somatostatin on gonadotropin release from the pituitary in vivo. Whereas cyclosomatostatin increased LH and FSH plasma levels at 2 h post-injection, octreotide decreased FSH levels after 2 h, but the LH levels remained unaffected. Overall, our findings provide important insights into the somatostatin system and its mechanisms of action, indicating a potential role in regulating growth and reproductive hormones. Further studies of the complex interplay between SST, its receptors, and reproductive hormones may advance reproductive control and management in cultured populations.

GnRH governs reproduction by regulating pituitary gonadotropins. Unlike most vertebrates, gnrh-/- zebrafish are fertile. To elucidate the role of the hypophysiotropic-Gnrh3 and other mechanisms regulating pituitary gonadotropes, we profiled the gene expression of all individual pituitary cells of wild-type and gnrh3-/- adult female zebrafish. The single-cell RNA sequencing showed that LH and FSH gonadotropes express the 2 gonadotropin beta subunits with a ratio of 140:1 (lhb:fshb) and 4:1 (fshb:lhb), respectively. Lh gonadotropes predominantly express genes encoding receptors for GnRH (gnrhr2), thyroid hormone, estrogen, and steroidogenic factor 1. No GnRH receptor transcript was enriched in FSH gonadotropes. Instead, cholecystokinin receptor-b and galanin receptor-1b transcripts were enriched in these cells. The loss of the Gnrh3 gene in gnrh3-/- zebrafish resulted in downregulation of fshb in LH gonadotropes and upregulation of pituitary hormones like TSH, GH, prolactin, and proopiomelanocortin-a. Likewise, targeted chemogenetic ablation of Gnrh3 neurons led to a decrease in the number of fshb+, lhb + and fshb+/lhb + cells. Our studies suggest that Gnrh3 directly acts on LH gonadotropes through Gnrhr2, but the outcome of this interaction is still unknown. Gnrh3 also regulates fshb expression in both gonadotropes, most likely via a non-GnRH receptor route. Altogether, while LH secretion and synthesis are likely regulated in a GnRH-independent manner, Gnrh3 seems to play a role in the cellular organization of the pituitary. Moreover, the coexpression of lhb and fshb in both gonadotropes provides a possible explanation as to why gnrh3-/- zebrafish are fertile.

With the growing demand for fish protein, more aquatic species are currently being introduced into aquaculture. A relatively new cultivar is the Australian barramundi (Lates calcarifer), a protandrous sequential hermaphrodite that takes several years to change from male to female. This delay prevents inter-generation breeding and hinders the establishment of sustainable genetic enhancement programs. In sequential hermaphrodites, sex change derives from the delicate balance between two steroids, 17β-estradiol (E2) and 11-ketotestosterone (11-KT). Here, we examined whether the generation time of fertile barramundi females could be shortened by injections of E2, which was administered in ethylene-vinyl-acetate (EVAc) implants at three doses of 0.5, 1, and 1.5 mg/kg BW; control fish received empty implants. An endocrine profile for E2 and 11KT and gonadal biopsies were examined throughout the study. Our results showed significant differences in E2 levels between the treated groups and the control 2 months after the last injection. The proportions of females in the 0.5, 1, and 1.5 mg/kg treatment groups were 80

The datasets include input files used for docking including the receptor models, docking grids and and ligand databases consisting of prepared ligand used in screening of potential allosteric modulators for carp FSHR and LHR. the original dataset were sourced from The compound libraries from https://enamine.net/compound-libraries and are free to access, downloaded and used as per the mentioned sites terms and conditions. The ligand Database given here are constructed and processed using the Phase module (Phase, Schrödinger, LLC, New York, NY, 2021.). The dataset also includes .pdb files of the docked ligands.

Serotonergic psychedelics are emerging therapeutics for psychiatric disorders, yet their underlying mechanisms of action in the brain remain largely elusive. Here, we developed a wide-field behavioral tracking system for larval zebrafish and investigated the effects of psilocybin, a psychedelic serotonin receptor agonist. Machine learning analyses of precise body kinematics identified latent behavioral states reflecting spontaneous exploration, visually-driven rapid swimming, and irregular swim patterns following stress exposure. Using this method, we found that acute psilocybin treatment has two behavioral effects: [i] facilitation of spontaneous exploration (“stimulatory”) and [ii] prevention of irregular swim patterns following stress exposure (“anxiolytic”). These effects differed from the effect of acute SSRI treatment and were rather similar to the effect of ketamine treatment. Neural activity imaging in the dorsal raphe nucleus suggested that psilocybin inhibits serotonergic neurons by activating local GABAergic neurons, consistent with psychedelic-induced suppression of serotonergic neurons in mammals. These findings pave the way for using larval zebrafish to elucidate neural mechanisms underlying the behavioral effects of serotonergic psychedelics.

In mammals, the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are macromolecules secreted during specific reproductive phases and display strict specificity towards their cognate receptors. However, fish gonadotropins (GTH) and their receptors (GTHR) display diverse species-specific expression patterns, secretion patterns, and intra- and interspecies cross-activation. To uncover the molecular basis of this diversity, we generated and analyzed 29 in-silico models of intra- and inter-species combinations of sturgeon, carp, tilapia, and human gonadotropins with piscine receptors and analyzed the resulting receptor activation and signal transduction of these GTHR-GTH complexes in-vitro. Our results suggest that unlike humans, the surface charge on piscine FSH/LH β-seatbelt and N107huLHCGR/K104hFSHR homologs does not necessarily determine binding specificity. Instead, sequence and structural variations allow piscine GTHs significant conformational flexibility when binding to the receptor extracellular domain, thereby enabling cross-activation. The resulting diversity may support various reproductive strategies in different environmental niches.

Life histories of oviparous species dictate high metabolic investment in the process of gonadal development leading to ovulation. In vertebrates, these two distinct processes are controlled by the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH), respectively. While it was suggested that a common secretagogue, gonadotropin-releasing hormone (GnRH), oversees both functions, the generation of loss-of-function fish challenged this view. Here, we reveal that the satiety hormone cholecystokinin (CCK) is the primary regulator of this axis in zebrafish. We found that FSH cells express a CCK receptor, and our findings demonstrate that mutating this receptor results in a severe hindrance to ovarian development. Additionally, it causes a complete shutdown of both gonadotropins secretion. Using in-vivo and ex-vivo calcium imaging of gonadotrophs, we show that GnRH predominantly activates LH cells, whereas FSH cells respond to CCK stimulation, designating CCK as the bona fide FSH secretagogue. These findings indicate that the control of gametogenesis in fish was placed under different neural circuits, that are gated by CCK.

Many fish species of interest in aquaculture, including the dusky grouper, Epinephelus marginatus, present reproductive dysfunctions due to confinement, which in turn affect the hypothalamus-pituitary-gonadal axis, which controls reproduction in vertebrates. The aim of this study is to use non-lethal tools to evaluate the reproductive activity of E. marginatus females in captivity. We measured the plasma profile of gonadal steroids and follicle-stimulating hormone (FSH) throughout 12 months and measured the oocyte's diameter. Additionally, we used human Chorionic Gonadotropin (hCG) and 17 beta-estradiol (E2) implants aiming to induce the vitellogenesis of non-vitellogenic females, and followed the plasma level of E2 and oocyte diameter. During the year, the concentration of the androgens testosterone (T) and 11-ketotestosterone (11-KT) did not change, while the plasma level of E2 significantly increased at the end of the cycle (September) compared with the first sampling month (October) in captivity. The plasma concentration of 17 alpha-hydroxyprogesterone (17 alpha-OHP) was significantly higher in November than in May, June, July, and August, and it was also higher in October compared to May and June. Plasma FSH levels were higher in January compared to April, June, July, August, and September, and were lower in April compared to October, December, February, and March. However, these hormonal changes did not result in changes in the oocyte's diameter, which remained unchanged, at the stage of primary growth throughout the annual cycle. The induction of vitellogenesis with hCG did not change E2 levels, while the higher dose of E2 implants increased the plasma level of this estrogen after 30 days, but this alteration did not reflect in the diameter of oocytes and vitellogenesis was not triggered in both treatments. The data suggest that one of the reasons for the impaired vitellogenesis in E. marginatus in captivity can be related with the plasma FSH level, which does not stimulate enough synthesis of E2 to promote vitellogenesis. Moreover, hCG or E2, at the concentration used in this experiment, were not able to trigger vitellogenesis in animals in captivity, reinforcing the fact that environmental cues, which are not available in captivity, are imperative to the function of the hypothalamus-pituitary-gonadal reproductive axis in E. marginatus.

In certain fish species, endogenous dopamine has a significant inhibitory effect on LH release and spawning induced by externally applied GnRH. Preovulatory LH surge and spawning in these fish species are caused by the simultaneous release of GnRH and exclusion of the dopaminergic inhibition. To induce spawning, a potent dopamine D2 receptor (DRD2) antagonist and a GnRH analog are concomitantly applied to fish with solid dopaminergic inhibition. However, the currently used dopamine antagonists are not approved by the FDA for veterinary use. This project was conducted in order to find a novel FDA-approved dopamine antagonist that will be efficient in spawning induction in fish species and will not be lethal to the fish. First, we studied the inhibitory effect of the novel dopamine antagonist, azaperone, on tilapia DRD2, transiently expressed in COS-7 cell line. Azaperone was more effective than metoclopramide in antagonizing the effect of quinpirole on taDRD2 both in terms of EC50 and maximal response. Next, we tested the ability of the combination of the dopamine antagonist and sGnRHa on GTH secretion, in adult tilapia. The novel dopamine antagonist significantly increased the GnRH-stimulated LH release in vivo, on both LH release and gene expression. We next aimed to test the novel combination in carp. The combination of the novel dopamine antagonist together with GnRHa, increased the release of estradiol and LH, in a dose-dependent manner. A higher hatching rate and spawning efficiency were observed than in the positive control that contained metoclopramide as a dopamine antagonist.

Vasoactive intestinal peptide (Vip) regulates luteinizing hormone (LH) release through the direct regulation of gonadotropin-releasing hormone (GnRH) neurons at the level of the brain in female rodents. However, little is known regarding the roles of Vip in teleost reproduction. Although GnRH is critical for fertility through the regulation of LH secretion in vertebrates, the exact role of the hypophysiotropic GnRH (GnRH3) in zebrafish is unclear since GnRH3 null fish are reproductively fertile. This phenomenon raises the possibility of a redundant regulatory pathway(s) for LH secretion in zebrafish. Here, we demonstrate that VipA (homologues of mammalian Vip) both inhibits and induces LH secretion in zebrafish. Despite the observation that VipA axons may reach the pituitary proximal pars distalis including LH cells, pituitary incubation with VipA in vitro, and intraperitoneal injection of VipA, did not induce LH secretion and lhβ mRNA expression in sexually mature females, respectively. On the other hand, intracerebroventricular administration of VipA augmented plasma LH levels in both wild-type and gnrh3-/- females at 1 hour posttreatment, with no observed changes in pituitary GnRH2 and GnRH3 contents and gnrh3 mRNA levels in the brains. While VipA’s manner of inhibition of LH secretion has yet to be explored, the stimulation seems to occur via a different pathway than GnRH3, dopamine, and 17β-estradiol in regulating LH secretion. The results indicate that VipA induces LH release possibly by acting with or through a non-GnRH factor(s), providing proof for the existence of functional redundancy of LH release in sexually mature female zebrafish.

Introduction: As in all vertebrates, reproduction in fish is regulated by gonadotrophin-releasing hormone (GnRH) control on gonadotrophic hormones (GtHs) activity. However, the neuroendocrine factors that promote GnRH and GtH activity are unknown. In Nile tilapia (Oreochromis niloticus), sexual activity and reproduction ability depend on social rank; only dominant males and females reproduce. Here, this characteristic of dominant fish allows us to compare brain and pituitary gene expression in animals that do and do not reproduce, aiming to reveal mechanisms that regulate reproduction. Methods: An extensive transcriptome analysis was performed, combining two sets of transcriptomes: a novel whole-brain and pituitary transcriptome of established dominant and subordinate males, together with a cell-specific transcriptome of luteinizing hormone (LH) and follicle-stimulating hormone cells. Pituitary incubation assay validated the direct effect of steroid application on chosen genes and GtH secretion. Results: In most dominant fish, as determined behaviorally, the gonadosomatic index was higher than in subordinate fish, and the leading upregulated pituitary genes were those coding for GtHs. In the brain, various neuropeptide genes, including isotocin, cholecystokinin, and MCH, were upregulated; these may be related to reproductive status through effects on behavior and feeding. In a STRING network analysis combining the two transcriptome sets, brain aromatase, highly expressed in LH cells, is the most central gene with the highest number of connections. In the pituitary incubation assay, testosterone and estradiol increased the secretion of LH and specific gene transcription. Conclusions: The close correlation between behavioral dominance and reproductive capacity in tilapia allows unraveling novel genes that may regulate the hypothalamic-pituitary-gonadal axis, highlighting aromatase as the main factor affecting the brain and pituitary in maintaining a sexually active organism.

The hypophysiotropic gonadotropin-releasing hormone (GnRH) and its neurons are crucial for vertebrate reproduction, primarily in regulating luteinizing hormone (LH) secretion and ovulation. However, in zebrafish, which lack GnRH1, and instead possess GnRH3 as the hypophysiotropic form, GnRH3 gene knockout did not affect reproduction. However, early-stage ablation of all GnRH3 neurons causes infertility in females, implicating GnRH3 neurons, rather than GnRH3 peptides in female reproduction. To determine the role of GnRH3 neurons in the reproduction of adult females, a Tg(gnrh3:Gal4ff; UAS:nfsb-mCherry) line was generated to facilitate a chemogenetic conditional ablation of GnRH3 neurons. Following ablation, there was a reduction of preoptic area GnRH3 neurons by an average of 85.3%, which was associated with reduced pituitary projections and gnrh3 mRNA levels. However, plasma LH levels were unaffected, and the ablated females displayed normal reproductive capacity. There was no correlation between the number of remaining GnRH3 neurons and reproductive performance. Though it is possible that the few remaining GnRH3 neurons can still induce an LH surge, our findings are consistent with the idea that GnRH and its neurons are likely dispensable for LH surge in zebrafish. Altogether, our results resurrected questions regarding the functional homology of the hypophysiotropic GnRH1 and GnRH3 in controlling ovulation.

ABSTR A C T Late maturation and large size at maturity are significant challenges when administering hormonal treatments in captive broodstock. Here, we report the potential of yeast, Pichia pastoris, as a vehicle to orally deliver a re-combinant gonadotropin-releasing hormone (rGnRH1) aimed at stimulating gonadal development in juvenile orange-spotted grouper, Epinephelus coioides. Two recombinant GnRH1 constructs were designed utilizing pPIC3.5, the yeast expression vector lacking a signal sequence, hence retaining the recombinant within the yeast. The first construct (rGnRH1_1xGAP) comprised of GnRH1 decapeptide and the GnRH-associated peptide (GAP) separated by a cleavage site. The second construct (rGnRH1_10x) consisted of ten GnRH1 decapeptides, each separated by a cleavage site. Expression of the two recombinant peptides was confirmed by mass spectrometry. ELISA for the GnRH analogue (GnRHa) was validated to determine the level of rGnRH1 from the yeast extracts. Parallelism between the serially diluted GnRHa and serially diluted extracts from recombinant yeast confirmed validity of the assay. A luciferase reporter assay showed stimulation of tilapia GnRH type 3 receptor by the yeast extract, suggesting biological activity in vitro. In a short term experiment, lyophilised yeast loaded in gelatine capsules and fed once to juvenile orange-spotted grouper resulted in a significantly higher plasma GnRH compared with the control. In a 4-week experiment, lyophilized yeast containing either rGnRH1_1xGAP or rGnRH1_10x was incorporated in fish pellets and fed daily at a dose of 1 mu g rGnRH1/kg fish body weight/day. The mean gonadosomatic index and oocyte diameter did not vary between the treated and control fish. The mean plasma levels of FSH, LH and GnRH in the rGnRH1-fed and control fish did not also vary significantly, however there were individuals in the rGnRH1-fed groups that had increased level of the hormones. Together with his-tological evidence showing oocyte development in a proportion of fish fed with the recombinant, results of the present study point to a biological activity in vivo of the rGnRH1 in yeast. With further refinement, this tech-nology has the potential to provide a non-invasive method for broodstock management of large, late-maturing species of fish and of small, delicate endangered species that require breeding in captivity.

African cichlids are well established models for studying social hierarchies in teleosts and elucidating the effects social dominance has on gene expression. Ascension in the social hierarchy has been found to increase plasma levels of steroid hormones, follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) as well as gonadosomatic index (GSI). Furthermore, the expression of genes related to gonadotropins and steroidogenesis and signaling along the brain-pituitary-gonad axis (BPG-axis) is affected by changes of an animal’s social status. In this study, we use RNA-sequencing to obtain an in-depth look at the transcriptomes of testes and pituitaries from dominant and subordinate male Nile tilapia living in long-term stable social hierarchies. This allows us to draw conclusions about factors along the brain-pituitary-gonad axis that are involved in maintaining dominance over weeks or even months. We identify a number of genes that are differentially regulated between dominant and subordinate males and show that in high-ranking fish this subset of genes is generally upregulated. Genes differentially expressed between the two social groups comprise growth factors, related binding proteins and receptors, components of Wnt-, Tgfβ- and retinoic acid-signaling pathway, gonadotropin signaling and steroidogenesis pathways. The latter is backed up by elevated levels of 11-ketotestosterone, testosterone and estradiol in dominant males. Luteinizing hormone (Lh) is found in higher concentration in the plasma of long-term dominant males than in subordinate animals. Our results both strengthen the existing models and propose new candidates for functional studies to expand our understanding of social phenomena in teleost fish.